Obesity has become a global epidemic afflicting both children and adults, and gradually spreading from the Western countries to the developing nations as well. It is now widely recognized that obesity is associated with, and is actually a major culprit in numerous comorbidities such as cardiovascular diseases (CVD), type 2 diabetes, hypertension, certain cancers, and sleep apnea/sleep-disordered breathing. As recently acknowledged by a joint American Heart Association and American Diabetes Association (AHA/ADA) statement, obesity is an independent risk factor for CVD, and CVD risks have also been documented in obese children. Obesity is associated with an increased risk of overall morbidity and mortality as well as reduced life expectancy. Indeed, not only obesity but also overweight are now listed as independent cardiovascular risk factors in the joint AHA/ADA call for the prevention of cardiovascular disease and diabetes.
This explicit inclusion of obesity in the first line of major risk confronts the health implications of the most dominant anthropometric change presently afflicting the human race world round, that of increasing visceral and total body fat mass. While finally acknowledged, the cardiovascular significance of this shift in body build is most likely still underestimated, as it apparently now affects even the youngest.
Obesity may comprise a more easily detectable hazard when associated with either some of its recognized sequels, such as the metabolic form of dyslipidemia (low high density lipoprotein cholesterol (HDLc), hypertriglyceridemia), diabetes and/or hypertension, or linked to co-existing risk factors transmitted genetically, independent of obesity. Such subtle interaction of overweight/obesity with cardiovascular disease may be also difficult to demonstrate because cardiovascular risk factors (excluding diabetes) have declined at all body mass index (BMI) levels but the decline appears to be greater at higher BMI levels. Additionally, although there is an increasing array of recognized adipocyte-derived factors that can negatively interact with the vasculature and/or glucose homeostasis, obesity may affect these factors differentially in a person-, gender-, age-, race- or ethnic-specific manner, thus potentially obscuring important adipose mass-dependent relationships. In other words, excess adipose tissue must not only be present, but also turned on to inflict damage.
With the exception of bariatric surgery, which can be presently offered to a limited number of subjects only, the lack of any truly effective treatment for obesity highlights the gravity of current prospects to control the obesity epidemic. Preventive measures have generally failed; effective public and political strategies to reshape lifestyle by proper nutrition and exercise so as to counteract the global obesity trends have not yet been formulated. Finally, the current generation of weight-reducing medications offers limited benefit, and indeed, despite more than a decade of use has failed to impact the global obesity challenge. Health service use and medical costs associated with obesity and related diseases have risen dramatically and are expected to continue to rise.
Hence, novel therapeutic strategies to combat fatness are needed. From a practical medical perspective, perhaps the most urgent need is to substantially reduce fat in individuals already afflicted with obesity, recognizing that excess fat, just like hypercholesterolemia, comprises a substantial health threat.
Apoptosis in Fat Cells
Increases in adipose tissue mass can result from an increase of the volume of adipocytes and/or from a rise in adipocyte number due to proliferation and differentiation of adipocyte precursor cells. These precursor cells, known as “preadipocytes”, extensively populate adipose tissue (Hauner et al., 1989).
Although reduction in fat tissue mass generally involves the loss of stored lipids by lipolytic processes, a reduction of the number of adipocytes may also be seen, especially in conditions where large amounts of fat are lost (Sjostrom et al., 1981). Indeed, contrary to former beliefs that weight loss results only from depletion of adipocyte fat stores and is not accompanied by change in fat cell number, there is now growing evidence that decreases in adipose tissue mass in humans could result from a loss of fat cells through programmed cell death (Prins et al., 1994; Prins et al., 1997; Prins et al., 1997b; Lloreta et al., 2002; Garg, 2000.). Fat cell apoptosis is now well recognized in patients with tumor cachexia and in human immunodeficiency virus (HIV) patients during treatment with protease inhibitors. Patients with acquired forms of lipodystrophy (Lawrence syndrome and Barraquer-Simons syndrome) show an immunologically mediated loss of fat cells, probably by apoptosis (Garg, 2000). In rodents, weight loss induced by starvation, streptozotocin-induced diabetes, or intracerebroventricular administration of leptin, results in apoptosis of fat cells (Geloen et al., 1989; Qian et al., 1998). In 3T3-L1 cells, apoptosis may be induced by serum deprivation or exposure to tumor necrosis factor-α (TNF-α) and HIV protease inhibitors (Gullicksen et al., 2003; Magun et al., 1998). Collectively these studies reinforce the emerging concept that adipocyte deletion by apoptosis is a significant contributor to the regulation of adipose tissue mass and to adipose tissue loss during weight reduction (Prins et al., 1997; Sorisky et al., 2000; Della-Fera et al., 2001). Further, a number of experimental ways have been proposed by which adipocyte apoptosis can be induced and through which it may potentially serve as a homeostatic mechanism to control fat cell number (Della-Fera et al., 2001; Scislowski et al., 1999; Margareto et al., 2000).
The current methods of induced apoptosis as a potential means to lower fat mass have been recently reviewed by Nelson-Dooley et al (Nelson-Dooley et al., 2005). In vivo adipocyte apoptosis has been successfully applied using leptin, ciliary neurotrophic factor (CNTF), beta-adrenergic agonists and conjugated linoleic acid (CLA) in rodents. However leptin treatment has generally failed in obesity other than leptin deficiency induced obesity due to leptin resistance (Hukshorn et al., 2002) and the overall response to CNTF-analog has been disappointing in terms of absolute weight loss (Ettinger et al., 2003) and the use of beta agonists in humans has been seriously hampered due to side effects (Connacher et al., 1992). Although adipocyte apoptosis can also be induced in vitro using TNF-α, its use is limited due to the inherent pro-inflammatory- and pro-atherosclerotic properties of this cytokine. (−)-Epigallocatechin gallate (EGCG) from Camellia sinensis and ajoene, a garlic product (Nelson-Dooley et al., 2005) were shown to precipitate apoptosis in vitro, but the adipocyte selectivity of these agents remains uncertain.
Expression-Inhibiting Oligonucleic Acids
The down regulation of specific gene expression in a cell can be effected by oligonucleic acids using techniques known as antisense therapy, RNA interference (RNAi), and enzymatic nucleic acid molecules.
Antisense therapy refers to the process of inactivating target DNA or mRNA sequences through the use of complementary DNA or RNA oligonucleic acids, thereby inhibiting gene transcription or translation. An antisense molecule can be single stranded, double stranded or triple helix.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in eukaryotic cells mediated by RNA fragments. The process of post-transcriptional gene silencing is thought to be an evolutionarily conserved defense mechanism used by cells to prevent the expression of foreign genes and is commonly shared by diverse organisms.
Other agents capable of down-regulating expression are enzymatic nucleic acid molecules such as DNAzymes and ribozymes, capable of specifically cleaving an mRNA transcript of interest. DNAzymes are single-stranded deoxyribonucleotides that can cleave both single- and double-stranded target sequences. Ribozymes are catalytic ribonucleic acid molecules that are increasingly being used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest.
Certain publications, e.g. U.S. Pat. No. 6,506,559 and PCT Pub. Nos. WO 01/75164 and WO 02/44321 relate to RNA interfering nucleic acids. International Patent Publication No. WO 2006/060454 teaches methods of designing small interfering RNAs, antisense polynucleotides, and other hybridizing nucleotides. US Patent Application Publication No. 20060217331 discloses chemically modified double stranded nucleic acid molecules for RNA interference. U.S. Pat. No. 6,506,559 discloses a process for inhibition of gene expression of a target gene in a cell using RNA having a region with double-stranded structure, wherein the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical.
The use of gene silencing agents for inducing apoptosis, for example in cancer therapy, has also been disclosed e.g. by WO 2005/083124, WO 2006/094406 and U.S. Patent Application Publication No. 2006/178330.
Adipose Targeting and Adipose-Specific Gene Expression
Administration of various compounds, including gene-silencing agents, to adipose cells and tissue has been contemplated. For example, WO 2004/029070, WO 03/104495, WO 03/104494 and WO 03/104477 to Marcusson et al. disclose methods for blocking adipocyte differentiation and triglyceride accumulation with inhibitors of DYRK4, Interleukin 12 p35, G-alpha-i3 or Transcription factor Dp-1, respectively. These applications suggest the use of various inhibitors including small molecules, antibodies, peptides (including dominant negative peptides) and nucleic acid agents, including ribozymes, inhibitory RNA molecules including siRNA molecules and antisense oligonucleotides.
It is becoming increasingly recognized that specific subpopulations of human adipocytes are the major pathogenic problem in human obesity. In essence, such cells are considered inflammatory and display a unique and specific gene expression profile (Jernas et al., 2006). For example, while both the Adiponectin and Leptin genes are specifically expressed in human adipocytes (Jernas et al., 2006; Mason et al., 1998; He et al., 1995), only the Leptin gene is further over-expressed as the cells become larger inflammatory adipocytes. It seems that different CCAAT/enhancer binding protein (C/EBP) family members bind to the adiponectin and leptin promoters in mature adipocytes and in large-cell adipocytes. It has been suggested, that the presence of an Sp1 binding site next to the C/EBP binding site in the leptin promoter induces preferential binding of the stimulatory C/EBP family member to the site (Mason et al., 1998; Salma et al., 2006). Nucleic acid constructs that would selectively or preferentially induce gene expression in adipose tissue, particularly constructs and vectors for inducing cell death preferentially in large-cell adipocytes, would be beneficial for controlling excess fat mass and pathological conditions associated therewith.
Platelet-Type 12-Lipoxygenase
Lipoxygenases (LOs) are dioxygenase enzymes that incorporate molecular oxygen into unsaturated fatty acids such as arachidonic acid and linoleic acid. These enzymes are named according to the carbon position (5, 12, or 15) at which they introduce oxygen. Arachidonate LOs and their products play an important role in mediating growth factor-induced tumor cell proliferation and appear to enhance the growth and migration of vascular smooth muscle cells (VSMCs). Some LO forms are also involved in LDL oxidation.
To date, several distinct LO genes have been structurally characterized. Three murine 12-LOs are currently recognized, including platelet 12(S)-LO leukocyte type, and an epidermal LO. Another LO, a porcine leukocyte type, has been isolated and cloned from porcine leukocytes, porcine pituitary cells, and bovine tracheal cells. The human platelet-type 12-LO has been cloned from human erythroleukemia cells and found primarily in human platelets, and was also identified in HEL (human erythroleukemia) cells, and umbilical vein endothelial cells. Platelet-type 12-LO metabolizes arachidonic acid to form exclusively 12(S)-hydroxyeicosatetraenoic acid (12-(S)HETE). There is also evidence for the presence of a leukocyte type of 12-LO in human adrenal glomerulosa cells. Additionally, an epidermal 12(R)-LO has recently been cloned from human skin.
Chemical inhibitors of 12-LO, which are not tissue- or cell type-specific, are known in the art. For example, U.S. Patent Application Publication No. 2002/0013368 discloses methods for prevention and/or treatment of diseases in which 5- and 12-LO activity contributes to the pathological condition, such as cancers and inflammation, by administration of 12-methyltetradecanoic acids, alone and in conjunction with other therapeutic compounds. The '368 publication does not teach or suggest inhibition of 12-LO expression or activity in adipocytes.
U.S. Pat. No. 5,861,268 discloses a method for inducing selective apoptosis of tumor cells which comprises contacting the tumor cells and the normal cells used as a control with an amount of a compound which inhibits 12-LO until apoptosis is induced in the tumor cells, wherein apoptosis is induced in the tumor cells without inducing apoptosis in the normal cells, wherein the compound is selected from the group consisting of: (1) a cyclic hydroxamic acid; (2) an aryl aliphatic acid; (3) nordihydro-guaiaretic acid, (NDGA); (4) N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP); (5) baicalein; and (6) an antisense segment of DNA which selectively binds to DNA encoding 12-lipoxygenase. The '268 patent does not disclose specific constructs and methods suitable for inducing apoptosis in adipocytes.
None of the prior art discloses that platelet-type 12-LO may be expressed in human adipocytes or pre-adipocytes and that it may mediate an anti-apoptotic function in these cells. Nowhere in the art is it suggested, taught or demonstrated that inhibition of 12-LO expression or activity may be effectively used to reduce fat cell mass, nor does the art disclose any constructs, compositions or methods for silencing 12-LO in fat cells in vivo. There exists an unmet medical need for effective and safe means for amelioration of conditions associated with excess fat cell mass and obesity.